Project Abstract/Summary The human body contains ~ 3.72 x 1013 cells and 200 different cell types. Generating the right number of cells overall and enough specialized cells is vital for building functional organs and tissues. How developing organisms generate and maintain cells with specialized functions and fates is a fundamental problem in biology. Asymmetric cell division is an evolutionary conserved mechanism to create sister cells with different fate. Cell fate differences can be implemented through the formation of unequal sized siblings. This form of asymmetric cell division ? here also referred to as physical asymmetry - is developmentally controlled since several metazoan cell types actively induce sibling cell size asymmetry or prevent the formation of sibling cells differing in their size. Physical asymmetric cell division can be induced by positioning the cleavage furrow off cell center. Since the predominant mechanism for cleavage furrow positioning originates from the mitotic spindle, spindle mispositioning or the generation of spindle asymmetry causes cleavage furrow formation off cell center. Alternatively, the dynamic behavior of the cell cortex ? regulated through DNA-derived, spindle- dependent or polarity cues ? can result in unequal cortical expansion to create different sized siblings. Naturally, these mechanisms can be applied in different combinations depending on developmental context and cell type. Here, we propose to use Drosophila larval neuroblasts to investigate molecular mechanisms regulating the dynamic behavior of the cell cortex during asymmetric cell division. Drosophila neuroblasts are neural stem cells in the fly, dividing asymmetrically by size and fate. We will use this model system to investigate how cell intrinsic polarity cues, acting in coordination with the cell cycle, control the localization and activity of actomyosin regulators to establish physical asymmetric cell division. We will also investigate how mechanical feedback loops influence spindle geometry and thus cleavage furrow positioning cues. We have implemented and developed a suite of novel and innovative tools to study these aspects in a developmental context in vivo. For instance, we are taking advantage of Drosophila?s superb genetic tractability and amenability for live cell imaging not available in other in vivo systems. We further utilize optogenetic approaches to manipulate the cell cortex with high spatiotemporal control. Our long-term goal is to understand the molecular, cellular and biophysical mechanisms underlying the generation of sibling cell size asymmetry. The respective size of sibling cells underlies stringent developmental control and has been implicated to regulate cell behavior and fate. Since sibling cell size asymmetry is ? and involved components are ? evolutionary conserved, this proposal will guide future studies in other phyla. The proposed research is also medically significant; several of the molecules under investigation have been implicated in cancer and investigating how sibling cell size asymmetry contributes to brain development is important to understand neurodevelopmental disorders such as microcephaly. This proposal will also have a strong impact in other fields. Tissue morphogenesis, organogenesis and stem cell behavior all depend on the correct spatiotemporal regulation of the cell cortex. Our research in conjunction with the tools and approaches we are developing will put us in a strong position to significantly contribute towards a mechanistic understanding of cortex-driven cell morphogenesis.